Mesenteric fat wrapping around the bowel wall, or ‘creeping fat’ (CF), is spatially associated with stricture formation in Crohn’s disease (CD). Intestinal muscularis propria (MP) smooth muscle cell hyperplasia is a major contributor to luminal narrowing in stricturing CD. We investigated the effect of mesenteric fat on intestinal muscularis propria thickness and smooth muscle cell hyperplasia in in vivo mouse models. We generated the FAT-ATTAC mouse, in which administration of the dimerizer AP20187 activates caspase-8, deleting adipocytes through apoptosis. All the mice were exposed to two-rounds of 1.5% dextran sodium sulfate (DSS) treatment to induce chronic colitis. Two strategies were employed to investigate the preventative and therapeutic role of fat deletion in chronic DSS colitis, consisting of AP20187 administration before DSS treatment and after the first round of DSS treatment, respectively. EchoMRI analysis confirmed a robust 70% reduction in body fat mass in FAT-ATTAC mice compared to baseline within 2 weeks following administration of the dimerizer AP20187. In preventative DSS colitis, there was no significant difference in weight loss, colon length, clinical colitis score, inflammation or fibrosis scores in the DSS treated animals (FAT-ATTAC compared to wildtype). As expected, in DSS treated WT mice the MP underlying the mesentery thickened, whereas FAT-ATTAC mice did not show any increase in MP thickening. Concordant with these findings, DSS treated WT animals showed an increase in proportion of Ki67+ cells in the intestinal MP underlying the mesentery and an increase in colonic smooth muscle cell (Sm22 and aSMA) gene expression, which was reduced in the DSS treated FAT-ATTAC mice. No difference of inflammation or fibrosis gene expression was observed when comparing DSS treated WT and FAT-ATTAC mice. In therapeutic DSS colitis, there was a significant difference in weight loss and clinical colitis score between DSS treated WT and FAT-ATTAC mice, largely due to fat deletion inducing weight loss in FAT-ATTAC mice. No difference of colon length, inflammation or fibrosis score was observed when comparing DSS treated WT and FAT-ATTAC mice. Importantly, the DSS treated FAT-ATTAC mice showed reduction in MP thickening and reduced proportion of Ki67+ cells in the intestinal MP underlying the mesentery. No significant difference of inflammation, fibrosis or smooth muscle cell associated gene expression was observed when comparing DSS treated WT and FAT-ATTAC mice. Deletion of fat in chronic DSS colitis reduced the MP thickness and smooth muscle cell proliferation underlying mesenteric fat. This suggests that presence of mesenteric fat is required for MP thickening underlying the mesentery in experimental colitis. These results point to CF as a novel contributor to stricture formation in CD.

Fibroblasts play a key role in stricture formation in Crohn’s disease (CD), but their mechanistic role in ulcerative colitis (UC) is unclear. Understanding fibrogenesis in UC requires a systems-level investigation to uncover new treatment targets. We studied full-thickness UC tissues to characterize fibroblast heterogeneity and function by generating the first single-cell RNA sequencing (scRNAseq) atlas of transmural UC bowel and providing proof of principle for therapeutic target validation. We performed scRNAseq of nine fresh full-thickness UC resections containing non-involved and inflamed segments as well as seven normal non-CD (non-CD or non-IBD) colon segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 64 tissue samples and 287,279 cells. We tested integrin a5b1 and avb1 heterodimers as potential anti-fibrotic therapeutic targets in UC with blocking antibody and small molecule respectively by using whole tissues, isolated intestinal cells, next-generation sequencing (NGS), and two mouse models. Our integrated dataset revealed fibroblast heterogeneity in inflamed UC with the majority of changes detected in the mucosa/submucosa, but also alterations in the muscle layer. Cell-cell interaction modeling revealed multiple fibroblast populations displaying a central signaling role in inflamed UC, including adventitial, MMP-expressing, and inflammatory fibroblasts. Fibroblasts emerged as major signal senders and interacted with each other through secretion of fibronectin (FN) and integrin signaling network. a5b1 and avb1 were fibroblast predominant integrins in UC, and inhibiting these heterodimers showed fibrosis resolution in our in vitro and in vivo experimental systems. Their function was validated by target expression, ECM deposition, migration, NGS and two animal models with a5b1 and avb1 blocking antibodies and small molecules. The first UC full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions. Integrins a5b1 and avb1 were validated as a potential therapeutic target. These results provide a new resource for a better understanding of UC fibrosis and open a potential therapeutic vulnerability of integrin heterodimers. Conflict of interest: Dr. Mukherjee, Pranab: No conflict of interest Chauhan, Gaurav: No conflict of interest Christensen, Stephen: No conflict of interest Khan, Afshin: None West, Gail: No conflict of interest Banerjee, Suhanti: No conflict of interest Chandra, Jyotsna: No conflict of interest Prasad, Ankita: No conflict of interest Czarnecki, Douglas: No conflict of interest Liu, Weiwei: No conflict of interest Larsen, Catherine: No conflict of interest Kirstein, Matthew: No conflict of interest Wong, Jamie: No conflict of interest Lu, Min: No conflict of interest Jain, Dhawal: Dhawal Jain is an employee and shareholder of Eli Lilly and Company Veisman, Ido: No conflict of interest Massey, William: No conflict of interest Wang, Yan: No conflict of interest Lal, Samir: No conflict of interest Fienman, Joshua: No conflict of interest Kravarik, Kellie: No conflict of interest Rieder, Florian: Personal Fees: Adiso, Adnovate, Agomab, Allergan, AbbVie, Arena, Astra Zeneca, Boehringer-Ingelheim, Celgene/BMS, Celltrion, CDISC, Celsius, Cowen, Ferring, Galapagos, Galmed, Genentech, Gilead, Gossamer, Granite, Guidepoint, Helmsley, Horizon Therapeutics, Image Analysis Limited, Index Pharma, Landos, Jannsen, Koutif, Mestag, Metacrine, Mopac, Morphic, Organovo, Origo, Palisade, Pfizer, Pliant, Prometheus Biosciences, Receptos, RedX, Roche, Samsung, Sanofi, Surmodics, Surrozen, Takeda, Techlab, Teva, Theravance, Thetis, UCB, Ysios, 89Bio

Intestinal fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM) in the bowel wall. Complications, such as strictures that require surgical intervention in a large proportion of patients, are considered an inevitable consequence of chronic inflammation in inflammatory bowel disease (IBD) and leads to severe complications. The study of intestinal fibrosis in IBD has been traditionally focused on the associated immune process, and the role of the ECM itself has been largely overlooked. More recent studies have now clearly demonstrated that ECM is not simply a passive bystander of inflammation-driven fibrosis but is instead an active participant in the initiation and progression of the fibrogenic process. In this narrative review, we first describe the composition and function of the ECM components under physiological and pathological conditions of the gut. Then, we review the alterations of the intestinal ECM in IBD-associated fibrosis and the impact of fibrotic ECM on intestinal biology and function. We next critically evaluate the existing experimental systems to study the intestinal ECM, both in vitro and in vivo. We conclude by discussing the unique challenges that still exist to better understand the role of the ECM in intestinal fibrosis, and its potential diagnostic and therapeutic implications.

INTRODUCTION:In inflammatory bowel diseases (IBD), human intestinal myofibroblasts (HIMF) gets activated due to chronic inflammation and start accumulating excessive extracellular matrix (ECM). ECM drives the significant clinical problem of intestinal fibrosis and stricture formation in Crohn's disease (CD) and Ulcerative colitis (UC) patients that require surgical intervention in a large group of the population. AREAS COVERED:In this review, we delineate the role of transglutaminase 2 (TG2), a matrix bound, calcium (Ca) dependent enzyme, as an important effector in the pathogenesis of chronic inflammatory diseases. We discuss the role of TG2 in fibrotic diseases with a focus on intestinal fibrosis and TG2 as a potential target for therapy of stricturing CD. This review additionally covers the progress in our mechanistic understanding of TG2 as a marker and driver for intestinal fibrosis and stricture formation in IBD patients. EXPERT OPINION:TG2 may play a central role in promoting inflammation independent progression of fibrosis, potentially explaining the lack of efficacy of traditional anti-inflammatory drugs in fibrotic diseases. The anti-fibrotic potential of TG2 specific inhibitors in stricturing Crohn's disease are just starting to be explored. Further investigations are needed to identify novel mechanisms for TG2 specific inhibitors in the intestine.

OBJECTIVE:Intestinal fibrosis is considered an inevitable consequence of chronic IBD, leading to stricture formation and need for surgery. During the process of fibrogenesis, extracellular matrix (ECM) components critically regulate the function of mesenchymal cells. We characterised the composition and function of ECM in fibrostenosing Crohn's disease (CD) and control tissues. DESIGN:Decellularised full-thickness intestinal tissue platforms were tested using three different protocols, and ECM composition in different tissue phenotypes was explored by proteomics and validated by quantitative PCR (qPCR) and immunohistochemistry. Primary human intestinal myofibroblasts (HIMFs) treated with milk fat globule-epidermal growth factor 8 (MFGE8) were evaluated regarding the mechanism of their antifibrotic response, and the action of MFGE8 was tested in two experimental intestinal fibrosis models. RESULTS:We established and validated an optimal decellularisation protocol for intestinal IBD tissues. Matrisome analysis revealed elevated MFGE8 expression in CD strictured (CDs) tissue, which was confirmed at the mRNA and protein levels. Treatment with MFGE8 inhibited ECM production in normal control HIMF but not CDs HIMF. Next-generation sequencing uncovered functionally relevant integrin-mediated signalling pathways, and blockade of integrin αvβ5 and focal adhesion kinase rendered HIMF non-responsive to MFGE8. MFGE8 prevented and reversed experimental intestinal fibrosis in vitro and in vivo. CONCLUSION:MFGE8 displays antifibrotic effects, and its administration may represent a future approach for prevention of IBD-induced intestinal strictures.

IntroductionIntestinal fibrosis is a common and serious complication of inflammatory bowel diseases (IBD) driving stricture formation in Crohn's disease patients and leading to submucosal damage in ulcerative colitis. Recent studies provided novel insights into the role of immune and nonimmune components in the pathogenesis of intestinal fibrosis. Those new findings may accelerate the development of anti-fibrotic treatment in IBD patients.Areas coveredThis review is designed to cover the recent progress in mechanistic research and therapeutic developments on intestinal fibrosis in IBD patients, including new cell clusters, cytokines, proteins, microbiota, creeping fat, and anti-fibrotic therapies.Expert opinionDue to the previously existing major obstacle of missing consensus on stricture definitions and the absence of clinical trial endpoints, testing of drugs with an anti-fibrotic mechanism is just starting in stricturing Crohn's disease (CD). A biomarker to stratify CD patients at diagnosis without any complications into at-risk populations for future strictures would be highly desirable. Further investigations are needed to identify novel mechanisms of fibrogenesis in the intestine that are targetable and ideally gut specific.

BACKGROUND & AIMS: In Crohn's disease, wrapping of mesenteric fat around the bowel wall, so-called "creeping fat," is highly associated with strictures. The strongest contributor to luminal narrowing in strictures is a thickening of the human intestinal muscularis propria (MP). We investigated creeping fat-derived factors and their effect on mechanisms of human intestinal MP smooth muscle cell (HIMC) hyperplasia. METHODS: Free fatty acids (FFAs) in creeping fat or non- creeping mesenteric fat organ cultures were measured via lipidomic mass spectrometry. Primary HIMCs were exposed to FFAs and cell proliferation was assessed. Intracellular FFA metabolism pathways and reactive oxygen species were functionally evaluated. Muscle thickness was investigated in dextran sodium sulfate colitis with small molecule inhibition of FFA transport and a novel fat deletion mouse model. RESULTS: Subserosal creeping fat is associated with a markedly thickened MP. Experimental deletion of mesenteric fat (FAT-ATTAC [fat apoptosis through targeted activation of caspase 8] mouse) reduced MP thickness. Human creeping fat-conditioned medium strongly up-regulated HIMC proliferation. Creeping fat released higher amounts of 5 long-chain FFAs, including palmitate. Inhibition of HIMC long-chain FFA metabolism or FFA uptake into mitochondria through carnitine palmitoyltransferase-1 reduced the palmitate-induced HIMC proliferation. Blockade of conversion of palmitate into phospholipids reduced HIMC proliferation. Prophylactic inhibition of carnitine palmitoyltransferase-1 in experimental dextran sodium sulfate colitis did not ameliorate inflammation, but reduced MP thickness. CONCLUSIONS: Creeping fat-released long-chain FFAs induce a selective proliferative response by HIMC. These results point to creeping fat as a novel contributor to stricture formation in Crohn's disease.

Background:Fibroblasts play a key role in stricture formation in Crohn's disease (CD) but understanding it's pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. Methods:We performed scRNAseq of 13 fresh full thickness CD resections containing non-involved, inflamed non-strictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next generation sequencing, proteomics and animal models. Results:Our integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and upregulated, and its pro-fibrotic function was validated by NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and two animal models of experimental colitis. Conclusion:A full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and opens potential therapeutic developments.

ObjectiveCreeping fat, the wrapping of mesenteric fat around the bowel wall, is a typical feature of Crohn’s disease, and is associated with stricture formation and bowel obstruction. How creeping fat forms is unknown, and we interrogated potential mechanisms using novel intestinal tissue and cell interaction systems.DesignTissues from normal, UC, non-strictured and strictured Crohn’s disease intestinal specimens were obtained. The muscularis propria matrisome was determined via proteomics. Mesenteric fat explants, primary human preadipocytes and adipocytes were used in multiple ex vivo and in vitro cell migration systems on muscularis propria muscle cell derived or native extracellular matrix. Functional experiments included integrin characterisation via flow cytometry and their inhibition with specific blocking antibodies and chemicals.ResultsCrohn’s disease muscularis propria cells produced an extracellular matrix scaffold which is in direct spatial and functional contact with the immediately overlaid creeping fat. The scaffold contained multiple proteins, but only fibronectin production was singularly upregulated by transforming growth factor-β1. The muscle cell-derived matrix triggered migration of preadipocytes out of mesenteric fat, fibronectin being the dominant factor responsible for their migration. Blockade of α5β1 on the preadipocyte surface inhibited their migration out of mesenteric fat and on 3D decellularised intestinal tissue extracellular matrix.ConclusionCrohn’s disease creeping fat appears to result from the migration of preadipocytes out of mesenteric fat and differentiation into adipocytes in response to an increased production of fibronectin by activated muscularis propria cells. These new mechanistic insights may lead to novel approaches for prevention of creeping fat-associated stricture formation.

Objective: Inflammatory bowel diseases (IBD) cause chronic intestinal damage and extracellular matrix (ECM) remodeling. The ECM may play an active role in inflammation by modulating immune cell functions, including cell adhesion, but this hypothesis has not been tested in IBD. Design: Primary human intestinal myofibroblast (HIMF)-derived ECM from IBD and controls, 3D decellularized colon or ECM molecule-coated scaffolds were tested for their adhesiveness for T cells. Matrisome was analysed via proteomics. Functional integrin blockade was used to investigate the underlying mechanism. Analysis of the pediatric Crohn's disease (CD) RISK inception cohort was used to explore an altered ECM gene expression as a potential predictor for a future complicated disease course. Results: HIMF-derived ECM and 3D decellularized colonic ECM from IBD bound more T cells compared to control. Control HIMFs exposed to the pro-inflammatory cytokines Iinterleukin-1β (IL-1β) and tumor necrosis factor (TNF) increased, and to transforming growth factor-β1 (TGF-β1) decreased ECM adhesiveness to T cells. Matrisome analysis of the HIMF-derived ECM revealed collagen VI as a major culprit for differences in T cell adhesion. Collagen VI knockdown in HIMF reduced adhesion T cell as did the blockage of integrin αvβ1. Elevated gene expression of collagen VI in biopsies of pediatric CD patients was linked to risk for future stricturing disease. Conclusion: HIMF-derived ECM in IBD binds a remarkably enhanced number of T cells, which is dependent on Collagen VI and integrin αvβ1. Collagen VI expression is a risk factor for a future complicated CD course. Blocking immune cells retention may represent a novel approach to treatment in IBD.

Mouse models are essential for investigation of underlying disease mechanisms that drive intestinal fibrosis, as well as assessment of potential therapeutic approaches to either prevent or resolve fibrosis. Here we describe several common mouse models of intestinal inflammation and fibrosis, including chemically driven colitis models, a bacterially triggered colitis model, and spontaneous intestinal inflammation in genetically susceptible mouse strains. Detailed protocols are provided for dextran sodium sulfate (DSS) colitis, 2,4,6-trinitro-benzene sulfonic acid (TNBS) colitis, adherent-invasive Escherichia coli (AIEC)-triggered colitis, the interleukin-10 knockout (IL-10KO) mouse model of spontaneous colitis, and the SAMP/YitFc model of spontaneous ileocolitis.